The invention relates in general to molecular separation technology utilizing microchip substrates and in particular to an apparatus and method for filling and cleaning microchannels and inlet ports of microchip substrates for use and reuse in the molecular separation of samples.
In the past ten years or so, parallel capillaries have been used extensively for molecular separations, such as by means of electrophoresis. Capillary electrophoresis has been used for the analysis of DNA and proteins, and for the separation of small ions, small molecules, bacteria, and viruses. Different separation media have been used in the capillaries including solutions, gels, and polymers. In each technique, the mobility of the target can be measured.
Capillaries have been applied both to DNA fragment length analysis and to DNA sequencing. The study of nucleotide sequences relies upon the high resolution separation of polynucleotide fragments. Each fragment in a family of fragments is tagged with fluorescent markers and the differences in the molecular migration in a capillary channel are observed. Fragments having differences of only a single base pair are routinely separated with fluorescent detection.
To increase the throughput, many capillaries can be used in parallel. Parallel capillary electrophoresis allows many samples to be analyzed simultaneously and can result in high throughput rates.
Recently, several groups have implemented capillary electrophoresis in microchannel formats (A. T. Wooley, G. F. Sensabaugh and R. A. Mathies, xe2x80x9cHigh-Speed DNA Genotyping Using Microfabricated Capillary Array Electrophoresis Chipsxe2x80x9d, Anal. Chem., 69:2181-2186 (1997); A. T. Woolley and R. A Mathies, Anal. Chem., 67:3676-3680 (1995); A. T. Woolley, P. C. Simpson, S. Liu, R. Johnston, G. F. Sensabaugh, A. N. Glazer, and R. A. Mathies, xe2x80x9cAdvances in Microfabricated Integrated DNA Analysis Systemsxe2x80x9d, HPCE98 (1998); P. C. Simpson, D. Roach, A. T. Woolley, T. Thorsen, R. Johnston, G. F. Sensabaugh, and R. A. Mathies, xe2x80x9cHigh-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplatesxe2x80x9d, Proc. Nat""l. Acad. Sci. USA, 95:2256-2261 (1998)). This approach uses microchannels etched or molded into a substrate as the separation channels in place of capillaries (R. M. McCormick, R. Nelson, M. G. Alonso-Amigo, D. J. Benvegnu and H. H. Hooper, xe2x80x9cMicrochannel electrophoretic separation of DNA in injection-molded plastic substratesxe2x80x9d, Anal. Chem. 69:2626-2630 (1997); U.S. Pat. No. 5,376,252, issued 1994 to B. Ekstrom, G. Jacobson, O. Ohman and H. Sjodin). The resulting device is commonly called a microchip, even though the physical size of the entire substrate can vary from microchip size, i.e. dimensions of a few millimeters on a side, to wafer size, i.e. dimensions similar to semiconductor wafers (10-20 centimeters diameter) to microchannels in 48 cm long xe2x80x9cmacrochipsxe2x80x9d (C. Davidson, J. Balch, L. Brewer, J. Kimbrough, S. Swierkowski, D. Nelson, R. Madabhushi, R. Pastrone, A. Lee, P. McCready, A. Adamson, R. Bruce, R. Mariella and A. Carrano, xe2x80x9cDevelopment of a Microchannel Based DNA Sequencerxe2x80x9d, DOE Human Genome Program Contractor-Grantee Workshop VI, Santa Fe, N.M. (1997)). The determining factors in microchip size are the complexity of microchannel routes and the lengths of the separation channels. The length of the channels must allow for sample input, sample migration and a measurement zone. The channels are typically of dimensions from 8 to 40 micrometers deep and 20 to 150 micrometers wide. The small channels resolve DNA fragments in significantly shorter times than capillaries with larger cross-sectional areas.
Beyond providing parallel capillaries, some advances in speed of analysis have been achieved by providing parallel sample wells and providing automated optical detectors and software analyzers.
In spite of these advances, fine separations are still a time consuming and labor intensive process, particularly when it comes to the preparation of the microchip substrate. Such preparations involve injection of separation media and solutions in the microchannels of the microchip substrate, filling inlet ports with solutions needed for analysis of samples, and cleaning the microchannels and inlet ports of the microchip substrate.
Prior art methods of filling and cleaning the microchannels and inlet ports of the microchip substrates involve a completely manual process. Matrix or other separation media is injected by syringe into common openings called anodes leading to the microchannels of the microchip substrate. The pressure of the matrix injected into the common anodes forces the old matrix out of the microchannels into a plurality of inlet ports on the microchip. Next, a pipette tip attached to a vacuum source is used to suction out solution or old matrix from the inlet ports one at a time. Sometimes, it is necessary to first add water to each of the individual inlet ports in order to dissolve any dry matrix so that it may be suctioned. Not only is this method time consuming, matrix in some of the microchannels may dry before all of the microchannels have been injected with new matrix. A dry matrix does not provide the electrically conductive path or the sieving characteristics necessary for proper separation of samples and is difficult to remove. Washing out the chip for storage involves a similar process in that each inlet port and all microchannels must be washed and dried individually. Although more microchannels present in the microchip substrate allow for more samples to be analyzed simultaneously, the preparation time of the entire microchip substrate increases.
An object of the invention was to devise an apparatus and method for efficient filling and cleaning of microchannels and inlet ports of microchip substrates for use in molecular separation and chemical analysis of samples. A further object was to automate the preparation of microchip substrates and to integrate the apparatus with the apparatus for the automatic handling and presentation of specimens into the microchips for parallel high throughput analysis in microchannels.
The above objects have been achieved with an apparatus for simultaneously cleaning and filling a large number of inlet ports and channels of a microchip substrate for use in molecular separations and chemical analysis of samples.
The microchip has macroscale inlet ports leading to the microchannels. The inlet ports are spaced apart to match the size and spacings of pipettors in an array of ganged pipettor tips.
The microchannels provide microscopic volumes, much less than a microliter, in which analysis is carried out. The instrument features a microchip handler, with relative motion of the microchip with respect to a pipettor, electrodes, and detector. In some instances the microchip is moved, while in other instances, the other components are moved. There is a sequence of automatic operations involving placing a sample-free microchip on a chuck, loading samples with a pipetting device into the microchip, contacting microchannels in the microchip with electrodes, injecting samples into the separation microchannels, running an electrophoretic separation, detecting and measuring the separation, and then removing the microchip. In a preferred embodiment, a microchip, pre-filled with matrix or other separation media but not sample, is held in a vacuum chuck which is movable with high precision on a first Y-axis track from a sample loading station to a sample analysis station.
In a preferred embodiment, the microchannels of the microchip are filled beforehand with matrix or other separation media that acts as a sieve to enhance sample separation. If the microchip substrate has already been used for molecular separation and chemical analysis and is in need of cleaning, an apparatus is used to clean and vacuum the inlet ports and to pump out the old matrix within the microchannels to the inlet ports for vacuuming before filling the microchip with new matrix. If the microchip substrate has not previously been used, the apparatus may be used to inject regeneration fluids, separation media or other solutions into the microchannels or solutions necessary for chemical analysis of samples into the inlet ports.
The apparatus has a manifold comprising six compartments. An upper chamber of the manifold is comprised of three compartments and a lower chamber of the manifold is comprised of three compartments. The three upper compartments each have opposed openings on each side for a total of six openings. Supply tubing which communicates with containers of solution is connected to the upper compartment openings. The three lower compartments each have opposed openings on each side for a total of six openings. Supply tubing which communicates with a vacuum source is connected to lower compartment openings. A plurality of openings on a lower surface of the upper chamber compartments and on lower surface of the bottom chamber compartments of the manifold are present for insertion of a tube-in-tube assembly.
The tube-in-tube assembly comprises a plurality of pressure tubes, surrounded by a plurality of vacuum tubes. The tube-in-tube assembly emerges from the manifold and engage the inlet ports of the microchip substrate. The apparatus also comprises a pressure injector manifold used to inject solution or matrix through the anodes or common openings of the microchip substrate leading into the microchannels of the microchip substrate. Old matrix is pumped out of the microchannels, if present, as new matrix or solution is pumped in. Wash solutions are pumped through the tube-in-tube assembly via supply tubing that communicates with the containers of solution. Solutions or matrix present in the inlet ports of the microchip substrate are vacuumed by the vacuum tubes of the tube-in-tube assembly which are connected to supply tubing that communicates with the vacuum source.
The apparatus may be automated and integrated with the apparatus for the automatic handling and presentation of specimens into the microchips for parallel high throughput analysis in microchannels.
At the sample loading station, samples can be loaded into the microchip by a multifunctional device, that includes a pipettor. The multifunctional device moves along a transverse X-axis gantry between the sample loading station on the first Y-axis track and tip and sample stations both on a second Y-axis track, parallel to the first Y-axis track. The second track can move pipette tips, reagent trays, microtiter trays containing samples, or other objects automatically into position for use by the multifunctional device. The multifunctional device, carried by the gantry, moves up and down on a Z-axis, perpendicular to the X and Y axes. Motion along all axes is driven by stepper motors so that precise and accurate positioning may be achieved. A servo motor or other actuator systems may be used for precise position control.
The multifunctional device contains a plurality of ganged pipettors, an individual pipettor, and a vacuuming line. The plurality of pipettors is ganged with spacings matching the well spacings on a microtiter plate. The same spacings are used for sample loading inlet ports on the microchip. In this manner, a multiple-channel pipettor can simultaneously load multiple samples into sample inlet ports.
The multifunctional device can be moved initially to the tip and sample stations on the second track where new pipette tips are applied to the ganged pipettors. The multifunctional device then moves on the gantry to pickup a tip guide and then moves back to the tip and sample stations on the second track. The second track can then be moved to a position where the ganged pipettors on the gantry can withdraw samples from a microtiter plate on the track. The multifunctional device then moves on the gantry to the sample loading station where it deposits the samples into sample inlet ports in the microchip on the first track. The multifunctional device moves back along the gantry first to release the tip guide and then to the tip and sample station where the used tips are discarded into a used tip tray that has been moved into position below the multifunctional device by the second track. The cycle of picking up tips, tip guide, and samples; delivering the sample to the microchip; and then parking the tip guide and discarding the used tips is repeated until the microchip has been completely loaded.
After the microchip has been loaded, it is moved to the sample analysis station on the first track below a sample analysis detector and raised to dock with the array of wire electrodes supported by a platform over the first track. The final position of the microchip places the microchannels in the focal plane of a detector at the sample analysis station. The detector preferably includes a scanning confocal laser microscope capable of detecting fluorescently tagged molecules during separation.
The electrical potential of the electrodes can be controlled to first move precise sub-microliter volumes of the samples from the loading wells into an injection region of the separation microchannels, and then to stimulate electromigration in the separation microchannels.
As the samples separate in the microchannels, a region of the microchip is monitored, typically by a scanning confocal laser microscope to detect the molecular separations. For DNA sequencing, four fluorescent markers are usually detected for forming four-color electropherograms of the separations. The four-color electropherograms can be processed to ultimately call the bases and determine the DNA sequence of the samples.